In the gastrointestinal (GI) tract, electroneutral Na+ absorption is regulated during the postprandial state as part of the neurohumoral response in digestion. Regulation of the intestinal brush border (BB) Na+/H+ exchanger, NHE3, accounts for most of the recognized digestion related changes in neutral Na+ absorption, as well as most of the inhibition of Na+ absorption that occurs in diarrheal diseases. NHE3 is regulated by changes in its plasma membrane versus intracellular location as a result of alterations in the rates of endocytosis and/or exocytosis. In the small intestine, elevation of intracellular calcium ([Ca2+]i) by carbachol (mimics postprandial changes due to cholinergic activation) inhibits NHE3 activity by 40% and similarly decreases NHE3 surface expression. However, the mechanisms responsible for [Ca2+]i regulated NHE3 trafficking in intestinal epithelial cells are not well understood. Therefore, the long-term objective of the current study is to establish the endocytic pathway that regulates NHE3 activity under elevated [Ca2+]i conditions which occur during normal digestion as well as during diarrheal diseases (e.g. rotavirus infection). The current proposal will attempt to identify whether clathrin-independent endocytosis contributes to the trafficking of an important regulator of electroneutral sodium absorption in intestinal epithelial cells. SPECIFIC AIMS: We propose to test the hypothesis that (1) inhibition of clathrin-mediated endocytosis (CME) prevents constitutive, but not carbachol-mediated, endocytosis of NHE3, and (2) carbachol-mediated endocytosis of NHE3 occurs through a lipid raft, activated Cdc42-dependent pathway that does not involve clathrin. Our concept that NHE3 endocytosis may occur through multiple endocytic pathways is novel and our current proposal will be the first to separate NHE3 endocytosis into clathrin-dependent and clathrin- independent pathways. We propose to use advanced imaging techniques including spinning disk confocal microscopy to characterize NHE3 endocytosis in four dimensions in live Caco-2BBe cells (polarized intestinal epithelial cell model) as well as shRNA knockdown technology to quantitate the critical components of clathrin- independent endocytosis, including Cdc42. SIGNIFICANCE: The use of advanced imaging will not only define how NHE3 endocytosis occurs but could also establish a new technique for studying the trafficking of other transport proteins, particularly those that exist in the apical domain of epithelial cells. Evaluating the mechanisms responsible for the regulation of NHE3 activity by elevated [Ca2+]i should contribute to an understanding of a mechanism responsible for diarrheal diseases that result from decreased NHE3 activity and reduced sodium absorption. Furthermore, establishing a role for clathrin-independent endocytosis in elevated [Ca2+]i regulation of intestinal transporters could provide important information in early events of transport protein regulation that could lead to alternate therapeutic strategies for treating acute diarrheal symptoms in diseases including rotavirus infection, inflammatory bowel disease, or congenital sodium diarrhea. PUBLIC HEALTH RELEVANCE: In the intestine, regulation of NHE3 activity accounts for most of the recognized changes in sodium absorption which occur during digestion as well as the inhibition of sodium absorption that occurs in diarrheal diseases. Elevated intracellular calcium-mediated inhibition of NHE3 activity may occur through an endocytosis pathway that is not clathrin-dependent;however, the mechanism for this regulation is not well understood. The current study will provide novel insights into understanding the trafficking of an important regulator of intestinal sodium absorption in digestion as well as in diarrheal diseases.